DESCRIPTION: Although a great deal of information has been gathered about iron metabolism in the past dozen years, one of the areas that remains a mystery is the mechanism by which iron is removed from transferrin in the endosome and transported across the membrane into the cytoplasm during the process of receptor-mediated endocytosis of the iron-transferrin complex. The Belgrade rat has a defect in iron metabolism that produces severe hypochromic, microcytic anemia. The Applicant was instrumental in demonstrating that the biochemical defect in these animals is defective incorporation of iron into developing red cell precursors. She has further located the defect to acidification of the endosome. She now plans to define precisely the defect in endosomal acidification in the Belgrade rat and to examine the biochemical basis of the problem. She has enlisted the collaborative support of Drs. Robert Murphy and Jonathan Glass. She and Dr. Murphy will use flow cytometric techniques to quantitate the acidification of the endosome using fluorescently- tagged transferrin as signal molecules. Dr. Glass has developed methods for the isolation of endosomes that contain transferrin from reticulocytes. In the preliminary data, Drs. Garrick and Glass have examined the acidification profile of endosomal vesicles isolated from Belgrade rat reticulocytes. These data demonstrate that (i) endosomes can be isolated from the reticulocytes of the Belgrade rat analagous to the isolations from rabbit reticulocytes, and (ii) the endosomes from the Belgrade rat have a defective profile of in vitro acidification. Acidification of endosomes depends, in part, on the proper activity of a group of vacuolar H+-ATPases (V-ATPases). Given the data that strongly points to endosomal acidification as a problem in the Belgrade rat, the P.I. plans to examine the molecular mechanism by purifying H+-ATPases from reticulocytes of Belgrade and normal rats, reconstituting these into liposomes, and determining whether defective acidification can be recapitulated in vitro. These experiments would provide a springboard to cloning the defective molecule, although this aspect of the study of the Belgrade defect is not included in the proposal.